In 2012, liver cancer was the second most common cause of cancer deaths worldwide. The incidence of hepatocellular cancer (HCC) is increasing, particularly in western societies where it is linked to the growing epidemic of obesity. Despite recent advances with targeted drugs like sorafenib, the prognosis for HCC patients with advanced disease remains poor, highlighting the urgent need for new therapies. The mammalian target of rapamycin (mTOR) protein kinase is a promising target in HCC, as the mTOR pathway has been shown to be upregulated in ~50% of HCCs, and the mTOR allosteric inhibitor rapamycin has been shown to inhibit tumor growth. However, the clinical use of rapamycin in HCC has had limited success, in agreement with our preclinical data using the rapamycin RAD001 in a mouse model of HCC. In the same study we found that RAD001 in combination with the dual PI3K/mTOR inhibitor BEZ235 synergistically inhibited mTOR signaling and growth of HCC tumors, which correlated with increased mitophagy. This finding is consistent with genetic data showing that autophagy in liver may act as a tumor suppressor. However, it was recently reported that mTOR inhibitors promote the survival of CD133+ HCC stem-like cells (HSCs), presumably through increased autophagy. Despite this observation, the biguanides phenformin and metformin, which activate AMPK, an upstream regulator of unc-51 like kinase (ULK1) and autophagy, are reported to selectively suppress cancer stem cells. In preliminary studies, we set out to compare the extent of autophagy induction in cells treated with RAD001/BEZ235 to those treated with phenformin, and the effects of the two treatments on the survival of HSCs. Unexpectedly, we found that phenformin not only failed to induce autophagy on its own, but inhibited RAD001/BEZ235 induced autophagy. Consistent with earlier reports, RAD001/BEZ235 treatment promoted the survival of HSCs in organoids derived from liver tumors, which was reversed by phenformin. These findings have led us to hypothesize that treatment of HCC with combined allosteric and ATP-site competitive inhibitors (mTOR paired inhibitors) effectively prevents bulk tumor growth, but promotes the autophagic-dependent survival of HSCs, which are sensitive to phenformin through the inhibition of autophagy, which we will test in two aims. In Aim 1, we will determine the mechanism by which phenformin inhibits autophagy and the relationship between autophagy and cellular energy status. We will determine the step(s) of autophagy inhibited by phenformin and the role of AMPK in regulating this response. In Aim 2, we will determine the therapeutic advantage of combining phenformin with mTOR paired inhibitors in HCC, when given together or sequentially. We will treat human HSCs and mouse HSC derived multi-population organoids with mTOR paired inhibitors with or without phenformin to test their therapeutic responsiveness. The ex vivo results will be compared to treatment of HCC tumor-bearing mice, with the goal of utilizing human HCC-derived organoids for future therapeutic applications.